Localization of Serine Racemase and Its Role in the Skin
Ran InoueYoko YoshihisaYosuke TojoChieko OkamuraYuzo YoshidaJiro KishimotoXinghua LuanMasahiko WatanabeMineyuki MizuguchiYuko NabeshimaKenji HamaseKenji MatsunagaTadamichi ShimizuHisashi Mori
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D-Serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)–type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier. D-Serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)–type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier. keratin 10 keratinocyte postnatal day 5 phosphate-buffered saline N-methyl-D-aspartate stratum corneum stratum granulosum serine racemase serine racemase–knockout transepidermal water loss transglutaminase 3 wild typeKeywords:
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Abstract The effect of Cu 2+ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole‐cell patch‐clamp and a fast perfusion system. Analysis of the Cu 2+ concentration‐response curve for inhibition of NMDA‐induced currents suggests that free Cu 2+ directly inhibits NMDA receptors with an IC 50 of 0.27 μM. Cu 2+ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA‐Cu 2+ and glycine‐Cu 2+ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu 2+ (10–100 μM) of responses to 10 μM NMDA was essentially voltage‐independent. The onset of inhibition by 100 μM Cu 2+ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU 2+ acts as a non‐competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu 2+ to NMDA receptors, calculated from the rate of onset of block by Cu 2+ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cu z+ interacts preferentially with agonist‐bound receptors. Our results show that Cu 2+ is a potent inhibitor of NMDA receptor‐mediated responses.
Long-term depression
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硬膜外鎮痛法は周術期や慢性･急性疼痛の管理に有意義である. 使用される薬剤はモルヒネが主流であるが, さまざまな薬剤が試用され, その評価もさまざまである. 硬膜外投与された薬剤は拡散して脊髄に作用するため, 血液脳関門は関与しない. 脊髄には多数のμおよびκ受容体が発現しているが, モルヒネなどのμ作動薬に比較してκ作動薬は副作用が少なく, 安全性が高い. それ故, 硬膜外κ作動薬は有意義な鎮痛法であり, 全身の鎮痛 (頭部･顔面に対しては頸部硬膜外鎮痛で可能) に応用できる. 一方, NMDA拮抗薬は, 痛覚過敏や中枢性感作などの疼痛の促通･増幅を抑制し (鎮痛とは異なる) , オピオイドとの相乗作用や耐性･依存性抑制作用がある (オピオイドの補助薬としての適応) . しかし, 一次求心性インパルスをブロックしないためNMDA拮抗薬に脊髄性鎮痛作用は期待できない. 全身投与によるNMDA拮抗薬は上位中枢神経系の統合機序に影響すると考えられ, 中枢痛･視床痛などには効果が期待できる. しかし, 硬膜外NMDA拮抗薬の効果は脊髄に限られるので, 全身投与に優る効果は期待できない. ケタミンについて考えると, この薬剤は作用特異性が低く, 多彩な効果が期待できる. これにより, 大量投与で全身麻酔作用が, 少量投与で鎮痛作用が現われると考えられる.
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N-methyl-D-aspartate(NMDA) 수용체는 중추신경계의 대표적 흥분성 신경전달물질인 glutamate의 수용체 중 하나로, NMDA 수용체 기본단위들의 발현 상태나 위치, 활동 정도에 따라서 수많은 신경학적, 정신적인 상태가 결정 될 수 있다. 많은 신경정신 질환들은 시냅스의 결함, 특히 NMDA수용체의 기능 장애가 그 원인으로 지목되고 있다. NMDA 수용체의 과잉활성뿐만 아니라 활동저하 역시 신경학적인 변화를 초래할 수 있기 때문에 NMDA 수용체 길항제와 부분효능제가 중추신경계 관련 질환의 치료제로 사용되고 있다. Glutamate의 증가는 뇌 관련 질환을 일으키거나 신경세포의 사멸을 초래하기 때문에 NMDA 수용체의 길항제가 이와 관련된 치료제가 될 가능성이 있다. 또한 지속적인 glutamate의 증가는 파킨슨병이나 알츠하이머병과 같은 퇴행성 신경질환을 야기하기도 한다. 동물 실험에서 NMDA 수용체 길항제가 허혈성 뇌 손상이나 외상적인 손상에 대해 신경보호 효과를 보여주는 것이 이를 뒷받침해준다. 그러나 NMDA 수용체를 구성하는 기본단위들에 대한 선택적인 약물들이 아닌 광범위한 길항제들은 부작용을 일으키고 치료효과가 높지 않다는 한계가 있다. 따라서 이러한 기본 단위들에 대한 선택적인 길항제에 대한 연구나 기본단위들의 발현 정도를 연구해야 할 필요성이 대두되고 있다. NMDA 수용체의 기본 단위는 세 가지 종류로 GluN1, GluN2(GluN2A, GluN2B, GluN2C, GluN2D), luN3(GluN3A,GluN3B)가 있으며, 전형적으로 GluN1과 GluN2의 결합으로 NMDA 수용체를 이룬다. NMDA 수용체 기본단위의 여러 조합은 중추신경계의 수용체가 다양성을 이루게 해주며 이러한 기본단위들의 조합과 발현 정도에 따라 NMDA 수용체의 기능이 결정 된다. 따라서 기본단위들의 조합이 NMDA 수용체의 조절능력의 중요한 요인이 되며 기본단위의 발현상태가 달라지면 NMDA 수용체의 기능이상이 생길 수 있다. 많은 중추신경계질환들에서 이러한 기본단위의 변화로 인한 NMDA 수용체의 기능이상이 원인으로 지목되고 있다. 이러한 중추신경계 질환들에 대한 연구는 대부분 NMDA수용체의 기본단위 중 가장 많은 부분을 차지 하는 GluN2의 변화에 초점이 맞춰져 있다. GluN3A의 경우 조현병에 영향이 있다는 연구결과가 보고 되었으나, 신경정신학적 질병들에 대한 연구가 GluN2에 비해 많이 진행되고 있지 않다.GRIN3A는 GluN3A를 암호화하는 유전자로서 GluN3A는 다른 NMDA 수용체의 기본단위들 보다 적은 부분을 차지하지만 시냅스의 발달에 관여하는 것으로 보고되고 있고, GluN3A의 증가는 NMDA 수용체의 기능을 저하시키는 것으로 알려져있다. 본 연구진은 사전 연구에서 GRIN3A가 주의력 결핍 과잉행동 장애 동물 모델인 본태성 고혈압 쥐에서 현저히 낮게 발현되는 것을 확인하였다.이를 통해 GluN3A가 신경정신학적 관련 질병에 기여 할 수 있다고 예측되는바 본 연구에서는 GRIN3A의 유전자 조작 마우스를 사용하여 GluN3A가 신경정신계에 어떤 영향을 미치는지 밝히고자 하였다.N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play a significant role in synaptic plasticity, learning, memory, and other behaviors. NMDARs are composed of various subunits and display unique properties depending on their subunit composition. The GluN3A subunit is thought to suppress NMDAR activity, however its exact function has not been well studied. Thus, we designed a transgenic mice overexpressing GRIN3A to assess the role of this gene on various behaviors. GRIN3A-overexpressing mice were subjected to an array of behavioral experiments including locomotor activity test, rota-rod test, elevated plus-maze (EPM) test, and Y-maze test. GRIN3A-overexpressing mice displayed hypoactivity but no effects on the rota-rod test, EPM test and Y-maze test. The GRIN3A-overexpressing mice that we have created can be utilized in elucidating the role of the GRIN3A gene and its product, the GluN3A subunit of NMDARs, on various behaviors associated with neuropsychiatric disorders, such as ADHD, and in finding possible therapeutic targets for these disorders.
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Ως κρίσιμη περίοδο ορίζουμε μια πολύ συγκεκριμένη χρονική περίοδο κατά την οποία ερεθίσματα από το περιβάλλον ρυθμίζουν την ανάπτυξη των νευρωνικών δικτύων του εγκεφάλου και δημιουργούν μόνιμες αλλαγές. Μια κρίσιμη περίοδος στο οπτικό σύστημα που έχει μελετηθεί εκτενώς αφορά στις μεταγεννητικές ημέρες 19 έως 32 κατά τις οποίες εξωγενή οπτικά ερεθίσματα είναι απαραίτητα για τη δημιουργία οφθαλμικών στηλών στο φλοιό για τον κάθε οφθαλμό. Έως τώρα όμως, καμία μελέτη δεν έχει εξετάσει συγκεκριμένες μεταγεννητικές περιόδους ανάπτυξης που να επηρεάζουν συγκεκριμένες γνωστικές λειτουργίες κατά την ενηλικίωση. Στόχος αυτής της μελέτης είναι να διερευνηθεί η ύπαρξη κρίσιμων περιόδων για την ανάπτυξη διαφορετικών γνωστικών λειτουργιών οι οποίες βασίζονται στη λειτουργία του προμετωπιαίου φλοιού και του ιπποκάμπου. Η μελέτη εστιάζει στη δεύτερη μεταγεννητική εβδομάδα, κατά τη διάρκεια της οποίας συμβαίνουν έντονες αλλαγές τόσο στον προμετωπιαίο φλοιό όσο και στον ιππόκαμπο, συμπεριλαμβανομένων και αλλαγών στα επίπεδα έκφρασης των υπομονάδων των NMDA-υποδοχέων του γλουταμινικού οξέος που υποστηρίζουν μηχανισμούς πλαστικότητας στον εγκέφαλο. Χρησιμοποιήθηκε το μοντέλο νεογνικής χορήγησης MK-801 ώστε να προκληθεί αναστολή της λειτουργίας των NMDA-υποδοχέων σε διακριτές χρονικές περιόδους κατά τη διάρκεια της δεύτερης μεταγεννητικής εβδομάδας: α) στο τέλος της 2ης μεταγεννητικής εβδομάδας, β) σε όλη τη διάρκεια της 2ης μεταγεννητικής εβδομάδας και γ) στην αρχή 2ης μεταγεννητικής εβδομάδας. Το νεογνικό μοντέλο ΜΚ-801 όπου αποκλείονται οι NMDA-υποδοχείς προς το τέλος της δεύτερης μεταγεννητικής εβδομάδας (p11-p15) παρουσιάζει ελλείμματα στη δοκιμασία αναγνώρισης χρονικής σειράς αντικειμένου, στη δοκιμασία απόσβεσης μνήμης φόβου, στη δοκιμασία κοινωνικότητας και στη δοκιμασία μνήμης κοινωνικότητας. Παράλληλα αυτό το μοντέλο χαρακτηρίζεται από μειωμένη αυθόρμητη δραστηριότητα σε προμετωπιαίο φλοιό και ιππόκαμπο. Ως προς το συγχρονισμό των νευρωνικών κυκλωμάτων, παρουσιάζεται μείωση των υψηλών γάμμα ταλαντώσεων στον προμετωπιαίο φλοιό και αύξηση των γάμμα ταλαντώσεων στον ιππόκαμπο. Ο αποκλεισμός των NMDA-υποδοχέων λοιπόν στο τέλος της 2ης μεταγεννητικής εβδομάδας επηρεάζει κατά κύριο λόγο τις γνωστικές λειτουργίες που βασίζονται κυρίως στη λειτουργία του προμετωπιαίου φλοιού. Όσον αφορά στο νεογνικό μοντέλο χορήγησης ΜΚ-801 σε όλη τη δεύτερη μεταγεννητική εβδομάδα (p7-p14), αυτό παρουσιάζει ελλείμματα στις δοκιμασίες αναγνώρισης χρονικής σειράς αντικειμένου, νέας θέσης αντικειμένου και νέου αντικειμένου. Επίσης παρουσιάζει ελλείμματα στη δοκιμασία απόσβεσης μνήμης φόβου και στη δοκιμασία κοινωνικότητας. Στη δοκιμασία μνήμης κοινωνικότητας έλλειμμα παρουσιάζουν μόνο οι θηλυκοί μύες. Τα παραπάνω ελλείμματα συνοδεύονται από μειωμένο αριθμό κυττάρων που εκφράζουν παρβαλβουμίνη στον προμετωπιαίο φλοιό, αλλά όχι στον ιππόκαμπο, ενώ παράλληλα το εν λόγω μοντέλο χαρακτηρίζεται από μειωμένη αυθόρμητη δραστηριότητα στον προμετωπιαίο φλοιό και στον ιππόκαμπο. Στο συγχρονισμό των νευρωνικών κυκλωμάτων στον προμετωπιαίο φλοιό παρουσιάζεται αύξηση των ρυθμών θήτα, δέλτα και γάμμα ενώ παρατηρείται μείωση των υψηλών γάμμα ρυθμών. Αντιθέτως στον ιππόκαμπο παρατηρείται αύξηση σε όλους τους ρυθμούς. Ο αποκλεισμός των NMDA-υποδοχέων λοιπόν καθ’όλη τη διάρκεια της 2ης μεταγεννητικής εβδομάδας φαίνεται να επηρεάζει περισσότερο τις γνωστικές λειτουργίες που βασίζονται στη λειτουργία του προμετωπιαίου φλοιού και του ιπποκάμπου. Τέλος, το νεογνικό μοντέλο ΜΚ-801 στο οποίο γίνεται αποκλεισμός των NMDA-υποδοχέων στην αρχή της δεύτερης μεταγεννητικής εβδομάδας (p7-p11) χαρακτηρίζεται από ελλείματα στη δοκιμασία αναγνώρισης χρονικής σειράς αντικειμένου και νέου αντικειμένου καθώς και στη δοκιμασία κοινωνικότητας. Στη δοκιμασία αναγνώρισης νέας θέσης αντικειμένου, μόνο οι αρσενικοί μύες της ομάδας p7-p11 ΜΚ-801 παρουσιάζουν έλλειμμα. Επομένως, ο αποκλεισμός των NMDA-υποδοχέων αυτήν την περίοδο φαίνεται να επηρεάζει γνωστικές λειτουργίες που βασίζονται στη λειτουργία τόσο του προμετωπιαίου φλοιού όσο και του ιπποκάμπου. Η αυθόρμητη δραστηριότητα σε αυτό το μοντέλο μειώνεται μόνο στον προμετωπιαίο φλοιό, όπου φαίνεται να επηρεάζεται και ο συγχρονισμός των νευρωνικών κυκλωμάτων καθώς παρατηρείται μείωση των γάμμα και των υψηλών γαμμα ρυθμών. Τα παραπάνω αποτελέσματα οδηγούν στο συμπέρασμα πως υπάρχουν διαφορετικές κρίσιμες περίοδοι ανάπτυξης για τον προμετωπιαίο φλοιό και τον ιππόκαμπο, οι οποίες πιθανόν και να αλληλεπικαλύπτονται, καθώς επέμβαση σε περιορισμένα χρονικά παράθυρα κατά τη δεύτερη μεταγεννητική εβδομάδα οδηγεί στην εμφάνιση δυσλειτουργιών του προμετωπιαίου φλοιού και του ιπποκάμπου με τρόπο εξαρτώμενο από τη χρονική περίοδο της έκθεσης. Μελλοντική διαλεύκανση του μηχανισμού θα οδηγήσει σε πιθανούς αποτελεσματικούς θεραπευτικούς στόχους νευροαναπτυξιακών διαταραχών όπως η Σχιζοφρένεια.
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To identify the structure and the function of NMDA receptors, to understand the modulatory mechanism of some endogenous and exogenous compounds on NMDA receptors, and to provide theoretical basis for developing new drugs that modulate NMDA receptors.A total of 24 originally identified articles were selected.A total of 24 articles were selected from several hundred original articles or reviews. The content of selected articles are in accordance with our purpose and the authors are authorized scientists in the study on NMDA receptors.After careful review of the selected papers, the meaningful results and conclusions were extracted using scientific criteria and our experience in the research of NMDA receptors.NMDA receptor contains at least five subunits. They were designated as the NR1 (sigma 1), NR2A (epsilon 1), NR2B (epsilon 2), NR2C (epsilon 3), and NR2D (epsilon 4). A unique feature of NMDA receptor is the requirement for both glutamate and the co-against glycine for the efficient gating. NMDA receptor is modulated by a number of endogenous and exogenous compounds. Mg2+ not only blocks the NMDA channel in a voltage-dependent manner but also potentiates NMDA-induced responses at positive membrane potentials. Na+, K+ and Ca2+ not only pass through the NMDA receptor channel but also modulate the activity of NMDA receptors. Zn2+ blocks the NMDA current in a noncompetitive and a voltage-independent manner. It has been demonstrated that polyamines do not directly activate NMDA receptors, but instead act to potentiate or inhibit glutamate-mediated responses. The activity of NMDA receptors is also strikingly sensitive to the changes in H+ concentration, and partially inhibited by the ambient concentration of H+ under physiological conditions. regulated by ion channels that are permeable to Ca2+, Na+, K+ and are sensitive to voltage-dependent Mg2+ block. This channel complex contributes to excitatory synaptic transmission at sites throughout the brain and the spinal cord, and is modulated by a number of endogenous and exogenous compounds. NMDA receptors play a key role in wide range of physiologic and pathologic processes. Five NMDA receptor subunits have now been characterized in both rat and mouse brain.
Long-term depression
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The N-methyl-D-aspartate (NMDA) receptor consists of a recognition site for NMDA, a cation-selective ion channel, and binding sites for glycine, Zn2+, and phencyclidine-like compounds. In addition, the channel can be blocked by Mg2+. We have studied the NMDA receptor using the potent and specific phencyclidine-like compound [3H]MK-801. Drugs that bind to the NMDA, glycine, Zn2+, and Mg2+ recognition sites profoundly affect both the association and the dissociation rate of [3H]MK-801. NMDA-like agonists, glycine, and Mg2+ all increase the rates of association and dissociation of [3H]MK-801, whereas the NMDA antagonists AP5 and Zn2+ decrease these rates. These data allow the construction of a model of drug interaction at the NMDA receptor that is based on the binding of MK-801 within the NMDA-operated channel. Using this model it is possible to clearly distinguish between drug action at any of the five binding sites proposed.
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In cerebellar granule Cell cultures, glutamate and N‐methyl‐D‐aspartate (NMDA) caused either neurotoxic or trophic effects, depending on the developmental stage of the neurones. Ethanol (100 mM) partly inhibited delayed neurotoxicity induced by the excitatory amino acids (25μM glutamate for 15 min or 100/tM NMDA for 30 min) assessed 24 h after the incubations in mature cultures in the absence of Mg 2t . Glycine (5 μM) potentiated the toxicity of glutamate and the ethanol inhibition, and was routinely added in these experiments. The viability of neurones in the presence of 25 mM K + and 0.8 mM Mg 2t was not impaired when maintained in 40–50 mM ethanol for the whole culture period of 7 days. However, ethanol almost completely inhibited the trophic effects of NMDA on developing cultures in 12.5 mM Kμ0.8 mM Mg 2+ medium. Glutamate (25 μM) and NMDA (100μM) potently induced 45 Ca 2+ uptake by granule cells from day 2 in vitro onward. Sixty‐five per cent of the 15‐min 45 Ca 2f influx induced by glutamate and 80% of that induced by NMDA were inhibited by ethanol (100 mM). MK‐801 (a non‐competitive antagonist of NMDA receptors; 100 nM) completely inhibited the toxic and trophic actions of glutamate and NMDA, as well as the 45 Ca 2+ influx induced by NMDA, but only 80% of the 45 Ca 2f influx induced by glutamate. These results show that the toxic and trophic actions of glutamate are mediated mainly by Ca 2+ influx through NMDA receptors. Both of these actions and the underlying Ca 2+ influx are significantly inhibited by ethanol at pharmacological concentrations (< 100 mM), although the mechanisms of inhibition still need further study.
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Granule (geology)
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